Electrophoresis is one of the most widely used separation techniques in the biologically-related sciences. Molecular species such as peptides, proteins, and oligonucleotides are separated by causing them to migrate in a buffer solution under the influence of an electric field. This buffer solution normally is used in conjunction with a low to moderate concentration of an appropriate gelling agent such as agarose or polyacrylamide to minimize the occurrence of convective mixing.
Two primary separating mechanisms exist, separations based on differences in the effective charge of the analytes, and separations based on molecular size. The first of these mechanisms is limited to low or moderate molecular weight materials in the case of separations of oligonucleotides because in the high molecular weight range the effective charges of these materials become rather similar, making it difficult or impossible to separate them. In the case of proteins, charge and size can be used independently to achieve separations. Separations based on molecular size are generally referred to as molecular sieving and are carried out employing as the separating medium gel matrices having controlled pore sizes. In such separating systems, if the effective charges of the analytes are the same, the separation results from differences in the abilities of the different sized molecular species to penetrate through the gel matrix. Smaller molecules move relatively more quickly than larger ones through a gel of a given pore size. Oligonucleotides and medium to high molecular weight polypeptides and proteins are commonly separated by molecular sieving electrophoresis. In the case of proteinaceous materials, however, it is first generally necessary to modify the materials to be separated so that they all have the same effective charges. This is commonly done by employing an SDS-PAGE derivatization procedure, such as is discussed in "Gel Electrophoresis of Proteins," B. D. Hames and D. Rickwood, Eds., published by IRL Press, Oxford and Washington, D.C., 1981. The contents of this book are hereby incorporated herein by reference.
Sometimes it is desirable to separate proteinaceous materials under conditions which pose a minimal risk of denaturation. In such cases system additives such as urea and SDS are avoided, and the resulting separations are based on differences in both the molecular sizes and charges.
Most electrophoretic separations are today conducted in slabs or open beds. However, such separations are hard to automate or quantitate. Extremely high resolution separations of materials having different effective charges have been achieved by open tubular free-zone electrophoresis and isotachophoresis in narrow capillary tubes. In addition, bulk flow can be driven by electroosmosis to yield very sharp peaks. Such high efficiency open tubular electrophoresis has not generally been applied to the separation of medium to high molecular weight oligonucleotides, however, since these materials have very similar effective charges, as indicated above. In addition, open tubular electrophoresis does not provide size selectivity for proteinaceous materials. The questions thus arise whether electrophoresis on gel-containing microcapillaries can be employed to achieve high resolution separations of oligonucleotides, and whether the conventional procedure of SDS-PAGE can be accomplished on such microcapillaries. As demonstrated by the present disclosure, the answers to these questions are affirmative, although given its potential importance as a separating technique in the biological sciences, surprisingly little attention has been paid to microcapillary gel electrophoresis.
Hjerten has published an article in the Journal of Chromatography. 270, 1-6 (1983), entitled "High Performance Electrophoresis: The Electrophoretic Counterpart of High Performance Liquid Chromatography," in which he employs a polyacrylamide gel in tubes having inside dimensions of 50-300 micrometers, and wall thicknesses of 100-200 micrometers. However, this work suffers from limited efficiency and relatively poor performance due in part to the use of relatively wide bore capillaries, relatively low applied fields, high electrical currents, and insufficient suppression of electroendosmosis. He has also obtained U.S. Pat. No. 3,728,145, in which he discloses a method for coating the inner wall of a large bore tube with a neutral hydrophilic substance such as methyl cellulose or polyacrylamide to reduce electroendosmosis in free-zone electrophoresis in open tubes. In a later patent, No. 4,680,201, Hjerten discloses a method for coating the inner wall of a narrow bore capillary with a monomolecular polymeric coating of polyacrylamide bonded to the capillary wall by means of a bifunctional reagent. These capillaries are also open tubes to be used for free-zone electrophoresis. In the background section of the '201 patent, it is stated that coating the inner wall of the electrophoresis tube with a polymeric substance to reduce adsorption and electroendosmosis suffers from the drawbacks that the coating material must be renewed periodically since it apparently flushes out of the capillary during use, and that relatively thick layers necessary for complete coating cause zone deformation in electrophoresis. This '201 patent thus teaches away from coating the wall of a capillary with a polymeric substance applied as an adsorbed layer, and discloses instead that for suppression of electroendosmosis a monomolecular layer of polyacrylamide should be covalently attached to the tube wall.
The small amount of work in the field of gel electrophoresis in capillaries by researchers other than the present applicants has generally resulted in columns which were not highly stable and could not be subjected to sufficiently high electric fields to achieve high efficiencies and high resolution separations. Improved gel filled capillary columns for electrophoresis which provide superior stability, efficiency, and resolution would be of great value in bioanalytical chemistry.